Method of detecting an antibody in a liquid sample

ABSTRACT

The invention relates to a method of evaluating the immunological status of a subject comprising the steps of 1) determining the content of an antibody in a liquid sample from the subject using an immunoassay, wherein the reaction between the antibody of the sample and a ligand in the form of an antigen, an antibody or a hapten, the ligand being directed to the Fab region of the sample antibody, is carried out in the presence of other constituents of the sample to obtain a measurement  1 , 2) determining the content of an antibody in the liquid sample using an immunoassay, wherein the reaction between the antibody of the sample and a ligand in the form of an antigen, an antibody or a hapten, the ligand being directed to the Fab region of the sample antibody, is carried out in the absence of other constituents of the sample to obtain a measurement  2 , and 3) interrelating measurements  1  and  2  to express the interference and using the interference as a parameter for evaluating the immunological status of the subject.

The present application claims the priority benefits of Danish PatentApplication Ser. No. PA1998/00821 filed Jun. 24, 1998 and U.S.Provisional Application filed Jun. 25, 1998. The present inventionrelates to a method of detecting a specific antibody in a liquid sample.

PRIOR ART

WO 94/11734 describes a two-site immunoassay for an antibody using achemiluminescent label and a biotin bound ligand, said method comprisingthe steps of (a) mixing the liquid sample with a ligand antigen,antibody or hapten bound to biotin or a functional derivative thereof,an antibody directed against the antibody to be detected bound toparamagnetic particles and a chemiluminescent acridinium compound boundto avidin, streptavidin or a functional derivative thereof to form asolid phase complex, (b) magnetically separating the solid chase fromthe liquid phase, (c) initiating a chemiluminescent reaction, if any, inthe separated solid phase and (d) analysing the separated solid phasefor the presence of a chemiluminescent phase, which is indicative of thepresence of said antibody in the sample.

The prior art method is particularly suitable for measuring theconcentration of specific immunoglobulins in body fluids, such as aspecific immunoglobulin selected from the group of IgA, IgD, IgE, IgG,IgM and subclasses thereof.

The prior art method is also suitable for the detection andquantification of the total content of immunoglobulins in a class orsubclass, such as IgA, IgD, IgE, IgG, IgM and subclasses thereof.

In a preferred embodiment of the prior art method the formation of thesolid phase complex is effected in two steps, viz. a first step whereinthe sample is mixed with the biotin bound ligand antigen, antibody orhapten and the antibody bound to paramagnetic particles so as to form afirst solid phase complex, and a second step wherein thechemiluminescent acridinium compound is added to the first solid phasecomplex to form a second solid phase complex.

However, practical use of the prior art method has revealed the factthat in some applications of the method interference from other types ofimmunoglobulins and/or from other types of immunologically active serumcomponents than the one to be measured occurs leading to errors in theresults obtained.

The article “Capture assay for specific IgE”, V. Olivieri et al, Journalof Immunological Methods, 157 (1993) 65-72 discloses an assay for themeasurement of specific IgE in the serum of allergic patients usingmonoclonal anti-human IgE (coated to the wells of a microtiter plate)and biotinylated allergens in solution. In a single incubation IgE isbound to the solid phase through the Fc fragment and biotinylatedallergens react with their specific IgE Fab regions. In a second step,streptavidin-horseradish peroxidase conjugate is added to reveal theamount of biotin fixed on the solid phase. The article studies theinterference from high levels of non-specific IgE and fromallergen-specific IgG. The assay is found to be unaffected byallergen-specific IgG.

WO 98/16829 discloses an assay, wherein an anti-immnunoglobulin iscoupled to a microtiter plate well, which is then washed. The test serumis then added to the well to capture all of total targetedimmunoglobulin in the test sample. Then, the biological fluid sample isaspirated, and the microtiter plate is washed. The captured antibody onthe microtiter plate is then exposed to biotinylated antigen, the plateis washed, and streptavidin/alkaline phosphatase conjugate is added, andthe plate is washed again. The prior art assay may be used to monitorthe effect of treatment on H. Pylori infection with standard antibiotictherapy.

SUMMARY OF THE INVENTION

A first object of the invention is to provide a method of the type,wherein the targeted antibody is complexed to a Fc directed antibodycoupled to a solid particle and to a Fab directed ligand, which does notsuffer from the above explained drawback of interference from othercomponents of the test sample.

This first object is achieved with the method of the invention, theessential new feature of the invention being that an additional sequenceof separation and washing of the intermediate solid phase complexconsisting of particle with reactant antibody and sample antibody iscarried out prior to addition of ligand.

The method of the invention is based on the recognition that byintroducing such an additional sequence of separation and washing,potentially interfering excess material from the liquid sample as wellas potentially interfering excess component (ii) may be removed from themethod thus eliminating the risk of interference of the said factors insubsequent steps. It has surprisingly been found that the additionalsequence of separation and washing has reduced substantially and in somecircumstances eliminated the technical problem of interference betweendifferent types of immunologically active serum components.

The reduction of the interference obtained involves a number oftechnical advantages. In particular, it allows a more precisemeasurement of problematic sera having a difficult and unpredictableratio of mixture of antibodies. Furthermore, falsely positiveidentifications of an antibody may be avoided. Also, the reduction ofinterference allows precise measurements to be made in a wider antibodyconcentration range than with prior art methods.

A second object of the invention is to provide a method, which iscapable of evaluating and/or predicting the effect of a Specific AllergyVaccination (SAV).

This second object is obtained by the nature and the temporaldevelopment of the interference between different types ofimmunologically active serum components, e.g. antibodies, are used asparameters for evaluating/predicting the effect of a Specific AllergyVaccination treatment. Thus, it has surprisingly been found that thesaid parameters hold valuable information about the immunological statusof a person as well as the response of a person to a selected treatmentscheme.

A third object of the invention is to provide a method of evaluating theimmunological status of a subject.

The third object of the invention is obtained by the nature and thetemporal development of the interference between different types ofimmunologically active serum components, e.g. antibodies, are used as aparameter for evaluating the immunological status of a subject, inparticular evaluating/predicting the effect of allergy treatment,allergy vaccination treatment or Specific Allergy Vaccination treatment.Thus, it has surprisingly been found that the said parameter holdvaluable information about the immunological status of a person as wellas the response of a person to a selected treatment scheme.

A fourth object or the invention is to provide a method of evaluatingthe effect of allergy treatment of a subject.

The fourth object of the invention is obtained based on the recognitionthat the measurement obtained with the subassays 1, A and C, i.e. ameasurement, which is carried out in the presence of interfering factorsin the sample, is particular useful for evaluating the effect of allergytreatment, allergy vaccination treatment and Specific Allergy Treatment(SAV). Thus, the measurement obtained with this method is obtained underconditions, which correspond to in vivo conditions, and hence is a morephysiological and clinical relevant measurement for evaluating treatmenteffects.

First Aspect of the Invention

A preferred embodiment of the first aspect of the invention ischaracterized in that component (iii) of step (r′), (r) or (r″) andcomponent (iv) of step (s′), (s) or (s″), respectively, are added in oneoperation.

A first alternative embodiment of the first aspect of the invention ischaracterized in that the three-component solid phase complex formed instep (r′), (r) or (r″) prior to subjecting it to step (s′), (s) or (s″),respectively, is washed to remove non-complex bound compounds.

Second Aspect of the Invention

Specific allergy vaccination (SAV), formerly known as specificImmunotheraphy or Hyposensitization, has been used for the treatment ofType 1 IgE mediated allergic disease since the beginning of thiscentury.

The general benefits obtained through SAV are: a) reduction of allergicsymptoms and medicine consumption, b) improved tolerance towards theallergens in the eyes, nose and lungs and c) reduced skin reactivity(early and late phase reactions).

The basic mechanism behind the improvement obtained by SAV is unknown,but a number of common features can be extracted from the numerous SAVstudies performed in the last decades: 1) the amount of total IgE isunchanged during the treatment period, 2) the amount of allergenspecific IgE increases transiently during updosing, then it falls backto the initial (pretreatment) level, 3) the epitope specificity andaffinity of IgE remains unchanged, 4) allergen specific IgG, inparticularly IgG4, raises sharply during SAV, 5) a new Th0/1 response isapparently initiated and 6) the Th2 response seem unchanged. There is nocorrelation between the effect induced by SAV and the onset of specificIgG.

SAV induces a new immune response which matures during the treatmentperiod (Th0/1 T-cells are recruited, an allergen specific IgX (X may beA1, A2, G1, G2, G3, G4, M or D) is initiated). As the affinity (oramount/affinity) of the new antibody response, IgX, has matured, IgX maycompete efficiently with IgE for the allergen(s), inhibiting the“normal” Th2 based allergic response characterised by the cross-linkingof receptor bound IgE on the surface of mast-cells and basophils. Hence,clinical symptoms will gradually be reduced.

The present invention is based on the hypothesis that if one measuresthe amount or specific IgE in the presence and absence of competingcompounds (IgX and/or any other interfering substance), a measure forthe competitive capability of immune responses in the individual patientmay be calculated and this measure would correlate with an appropriateeffect parameter.

Most prior art “quantitative” IgE assays measure IgE in the absence ofcompeting substances. The present invention describes the measurement ofIgE in the presence and absence of any (serum originating) competingsubstance. Thus, the methods referred to in FIGS. 2a-c measure IgE inthe absence of competing agents, whereas the method defined in step(i′), (i), (i″), (y′), (y) and (y″), respectively, measure IgE in thepresence of competing agents.

It is believed that the mode of action of the method of the inventionmay be explained as follows: If SAV induces a response which competeswith IgE for the binding of the allergen it should be possible tomeasure the effect of the treatment by comparing IgE determined by theabove stated two methods, if the two methods produce the same result nointerfering substance has been induced and there is no effect.Furthermore, if the measurement of the latter method (in the presence ofcompeting substances) is lower than the measurement of the former method(in the absence of competing substances) a competing response has beenmounted and there is an effect of the treatment.

In the second aspect of the invention, two subassays are used in themethod, viz. subassays, wherein the reaction between sample antibody andallergen is effected in the absence (subassay (h′), (h), (h″), (x′), (x)and (x″)) and presence (subassay (i′), (i), (i″), (y′), (y) and (y″) ofthe other sample constituents, respectively.

In subassays (h) and (h″), the chemiluminescent label may be anacridinium compound.

A preferred embodiment of the subassays (h′), (h) and (h″) ischaracterized in that in step (a′), (a) or (a″) components (i), (ii) and(iii) are mixed in one operation (FIG. 3b).

A first alternative embodiment of the subassays (h′), (h) and (h″) ischaracterized in that in step (a′), (a) or (a″) components (i) and (ii)are mixed in a first operation and that component (iii) is added in asecond operation (FIG. 3c).

A second alternative subassays (h′), (h) and (h″) is characterized inthat in step (a′), (a) or (a″) components (i) and (iii) are mixed in afirst operation and that component (ii) is added in a second operation(FIG. 3a).

A preferred embodiment of the second aspect of the invention ischaracterized in that step (ia′), (ia), (ia″), (ya′), (ya) or (ya″) iscarried out by mixing components (i) and (ii), then adding component(iii), and finally adding component (iv), if added.

Another preferred embodiment of the second aspect of the invention ischaracterized in that step (ia′), (ia), (ia″), (ya′), (ya) or (ya″) iscarried out by mixing components (i), (ii) and (iii), and then addingcomponent (iv), if added.

A further preferred embodiment is characterized in that the comparisonof step (j′), (j), (j″), (z′), (z) or (Z″) is carried out by calculatingthe ratio of the measurements of the said two steps. Alternatively, thecomparison is carried out by calculating the difference between the twomeasurements.

Still a further embodiment of the invention is characterized in that thecomparison of step (j′), (j), (j″), (z′), (z) or (z″) is carried out ata number of points in time at the start of and during the treatmentperiod, and that any temporal change, which may be observed, is used asa basis for evaluating and/or predicting the effect of the treatment.

Third Aspect of the Invention

The third aspect of the invention is based on the same recognitions andhypothesis as the second aspect, the difference being that the thirdaspect is not limited to any specific immunoassay procedure. Also,according to the third aspect the assay may be used to predict theeffect of all types of allergy treatment.

The interrelating of the two measurements obtained may be carried out bycalculating the ratio of or the difference between the measurements.

The term “allergy treatment” means any treatment of allergy. The term“allergy vaccination treatment” means any vaccination treatment ofallergy. The term “Specific Vaccination Treatment (SAV)” is describedabove.

Definitions

In the present invention the expressions “the antibody of the sample”and “sample antibody” may mean a specific immunoglobulin, preferably aspecific immunoglobulin from the classes IgA, IgD, IgE, IgG, IgM andsubclasses thereof. In general, the said antibody may be any blood serumor plasma component, which is capable of interfering specifically withthe interaction between immunoglobulines and a ligand in the form of anantigen, an antibody or hapten, e.g. enzyme inhibitors and receptors.

The term “liquid sample” means any liquid or liquefied sample, includingsolutions, emulsions, dispersions and suspensions. The sample may be abiological fluid, such as blood, plasma, serum, urine, saliva and anyother fluid, which is excreted, secreted or transported within abiological organism.

The expression “ligand in the form of an antigen, an antibody or ahapten” may be any immunologically active substance. “Antigen” may be anallergen, e.g. pollen from trees, grass, weeds etc., mould allergens,allergens from acarids (mites) and animals, such as cat, dog, horse,cattle and bird, allergens of stinging insects and inhaled allergensoriginating from insects, and food allergens; “antibody” may be amonoclonal or polyclonal antibody, including recombinant and fragmentedantibodies; and “hapten” may be carbohydrate moieties or fragmentsthereof, enzyme inhibitors or drugs, e.g. penicillin or a derivativethereof.

The expression “labelled ligand” means any ligand comprising a labelledatom or part, e.g. a radioactive atom label.

The expressions “label compound” means any suitable label systemconventionally used in immunoassays comprising luminescent labels,chemiluminescent labels, enzyme labels, radioactivity labels,fluorescent labels, and absorbance labels.

The term “carrier” means any solid support, which may be used in animmunoassay, e.g. a microtiter plate, a particle, a tube, a sponge of apolymer material (matrix), etc.

The term “solid particle” means any particulate matter, which can besuspended in a liquid, e.g. glass beads, metal, e.g. iron particles,particles of polymer material, etc.

The separation of the solid phase complex from the liquid phase may,depending on the type of solid particle used, be carried out by i.a.magnetic separation, filtration, sedimentation, centrifugation,chromatography, column chromatography.

The term “solid paramagnetic particle” means any paramagnetic particle,which may be dispersed or suspended in a liquid medium, e.g. “Biomag”particles (iron oxide particles coated with amine terminated groups)sold by Advanced Magnetics Inc., U.S.A., and “Dynabeads” (iron oxidecovered with a polymer) sold by Dynal A. S., Norway.

The term “reactant antibody” means any antibody or other biospecificreagent capable of reacting with the sample antibody comprisingmonoclonal and polyclonal antibodies, including recombinant andfragmented antibodies, e.g. a monoclonal antibody, “MAb A5697-1A3(920325) supplied by BioInvent International AB, Sweden,“Protein A” or “Protein G” supplied by Sigma Chemical Company, SaintLouis, USA.

The chemiluminescent compound is preferably an acridinium compound, suchas N-hydroxy-succinimide dimethylacridiniumester (NHS-DMAE).Avidin/streptavidin and DMAE may be coupled according to the methods ofWeeks et al., Clinical Chem., 29, 1474-1479 (1983). Other examples ofchemiluminescent compounds suitable for use in the present invention areluminol, lucigenin and lophine.

In the following, the invention will be described in further detail withrespect to the figures, wherein

FIGS. 1a-b are diagrammatic representations or two embodiments (PriorArt Method A and B) of the prior art assay disclosed in WO 94/11734.

FIGS. 2a-c are diagrammatic representations of three preferred assays ofthe present invention.

FIGS. 3a-c are diagrammatic representations of three preferred assaysaccording to the first aspect of the invention.

FIGS. 4-6 show the chemiluminescence level as a function of theconcentration of specific IgE for Prior Art Method B, Method 1 of theinvention and Submethod 1 of the invention, respectively.

FIG. 7 shows the relative chemiluminescence level as a function of thecontent of interfering antibodies for Prior Art Method B, Method 1 ofthe invention and Submethod 1 of the invention.

FIGS. 8-12 show the calculated chemiluminescence level relative to theexpected level as a function of dilution factor for Prior Art Method Band Method 1 of the invention for four samples front patients subjectedto Specific Allergy Vaccination and one reference sample (FIG. 12).

FIGS. 13-14 show the relative response for patients subjected toSpecific Allergy Vaccination and placebo treatment at 0, 6 and 12 monthsfor Prior Art Method B and Method 1, respectively.

FIG. 15 show the ratio of responses obtained with Prior Art Method B toresponses obtained with Method 1.

FIGS. 16-18 show the relative IgE level at times 0, 6, 12 and 24 monthsfor the three clinical effect groups Effect, No Effect and Doubtful,respectively.

FIG. 19 shows the ratio of responses obtained with Method 1 to responsesobtained with Prior Art Method B as a function of time for various SAVtreatment doses.

FIG. 20 shows the ratio of mean responses obtained with Method 1 toresponses obtained with Prior Art Method B as a function of time forpatients with clinical effect and patients without clinical effect,respectively.

FIG. 21 shows the ratio of responses obtained with Method 1 to responsesobtained with Prior Art Method B as a function of time for individualpatients with clinical effect.

FIG. 22 shows the ratio of responses obtained with Method 1 to responsesobtained with Prior Art Method B as a function of time for individualpatients with no effect.

FIG. 1a shows the principle or one embodiment of the assay disclosed inWO 94/11734. In the assay the antibody to be detected (specific IgE) inthe sample (i) is mixed with a reactant antibody bound to a paramagneticparticle (ii) to form a two-component complex, which is incubated, andthen a biotinylated allergen (iii) is added to form a three-componentcomplex, which is incubated, and then a chemiluminescent acridiniumcompound bound to avidin/streptavidin (iv) is added to form afour-component complex, which is washed, and the chemiluminescence ofthe washed complex is then measured.

FIG. 1b shows another embodiment of the prior art assay, whereincomponents (i) and (iii) are mixed to form a two-component complex, andwherein components (ii) and (iv) are then added, and the resultingmixture is incubated to form a four-component complex, which is washedand subjected to chemiluminescence measurement.

In the assays of FIGS. 1a and 1 b all constituents of the sample,including any cross-reacting IgE antibodies and non-IgE antibodiesspecific to the allergen, are present in the subsequent reactionsleading to the formation of the four-component complex.

FIG. 2a shows a preferred embodiment of the second aspect of theinvention, wherein components (i) and (ii) are mixed and incubated toform a two-component complex, which is washed. The component (iii) isadded to form a three-component complex, after which component (iv) isadded to form a four-component complex, which is washed and subjected tochemiluminescence measurement.

FIG. 2b shows another preferred embodiment of the second aspect of theinvention, which corresponds to that shown in FIG. 2a except thatcomponents (iii) and (iv) are added in one operation.

FIG. 2c shows a further preferred embodiment of the second aspect of theinvention, which corresponds to that shown in FIG. 2a except that thethree-component complex formed is subjected to a washing step before theaddition of component (iv).

In the assays of FIGS. 2a, 2 b and 2 c the constituents of the sample,including any cross-reacting IgE antibodies and non-IgE antibodiesspecific to the allergen, are removed after reaction with component (ii)and are hence absent in the subsequent reaction steps leading to theformation of the four-component complex.

FIG. 3a shows a preferred embodiment of the subassays (h′), (h) and(h″), wherein components (i) and (iii) are mixed in a first operation toform a two-component complex, to which component (ii) is then added in asecond operation, and the resulting mixture is incubated to form athree-component complex, which is washed before adding component (iv) toform a four-component complex, which is then washed before subjecting itto chemiluminescence measurement.

FIG. 3b shows another preferred embodiment of subassays (h′), (h) and(h″), wherein the components (i), (ii) and (iii) are added in oneoperation to form a three-component complex, which is then washed beforeadding component (iv) to form a four-component complex, which is thenwashed before subjecting it to chemiluminescence measurement.

FIG. 3c shows a further preferred embodiment of subassays (h′), (h) and(h″), wherein components (i) and (ii) are mixed to form a two-componentcomplex, to which component (iii) is then added to form athree-component complex, which is washed before adding component (iv) toform a four-component complex, which is also washed before subjecting itto chemiluminescence measurement.

In the assays of FIGS. 3a, 3 b and 3 c the constituents of the sample,including any cross-reacting IgE antibodies and non-IgE antibodiesspecific to the allergen, are removed after reaction with components(ii) and (iii) and are hence absent in the subsequent reaction stepleading to the formation of the four-component complex.

In the following the invention will be described in further detail withreference to the examples.

PREPARATION OF REAGENTS

Biotinylated Dermatophagoides pteronyssinus

Dermatophagoides pteronyssinus extract, (ALK-ABELLÓ A/S, Hrsholm,Denmark) is biotinylated in the molar ratio of 30:1. Biotinamidocaproate N-hydroxysuccinimide ester (Biotin-XX-NHS, Clontech, USA)is dissolved in Dimethylformamide (Merck) to a final concentration of 25mg/ml. 55.4 μl of this solution of Biotin-XX-NHS is added to 1 ml of2.44 mg/ml Derinatophagoides pteronyssinus in 0.1 M NaHCO₃, pH 8.5. Thereagents are incubated for 15 minutes at 25° C. in an “end over end”mixer. The biotinylated extract is purified from unbound biotin by sizeexclusion chromatography on a PD10-column (Pharmacia). The fractioncontaining the allergens is collected. The biotinylated Dermatophagoidespteronyssinus is diluted with Phosphate buffered saline, pH 7.2 (PBS),containing 0.1% human serum albumin (Sigma) and 0.09% NaN₃ (Merck).

Biotinylated Alternaria alternata

Alternaria alternata extract, (ALK-ABELLÓ A/S, Hrsholm, Denmark) isbiotinylated in the molar ratio of 30:1. Biotin amidocaproateN-hydroxysuccinimide ester (Biotin-XX-NHS, Clontech, USA) is dissolvedin Dimethylformamide (Merck) to a final concentration of 25 mg/ml. 38.6μl of this solution of Biotin-XX-NHS is added to 1 ml of 1.7 mg/mlAlternaria alternata in 0.1 M NaHCO₃, pH 8.5. The reagents are incubatedfor 15 minutes at 25° C. in an “end over end” mixer. The biotinylatedextract is purified from unbound biotin by size exclusion chromatographyon a PD10-column (Pharmacia). The fraction containing the allergens iscollected. The biotinylated Alternaria alternata is diluted withPhosphate buffered saline, pH 7.2 (PBS), containing 0.1% human serumalbumin (Sigma) and 0.09% NaN₃ (Merck).

Biotinylated Betula verrucosa (Silver birch)

Betula verrucosa pollen extract, (ALK-ABELLÓ A/S, Hrsholm, Denmark) isbiotinylated in the molar ratio of 10:1. Biotin amidocaproateN-hydroxysuccininide ester (Biotin-XX-NHS, Clontech, USA) is dissolvedin Dimethylformamide (Merck) to a final concentration of 25 mg/ml. 33.5μl of this solution of biotin-XX-NHS is added to 1 ml of 4.4 mg/ml.Betula verrucosa in 0.1 M NaHCO₃, pH 8.5. The reagents are incubated for15 minutes at 25° C. in an “end over end” mixer. The biotinylatedextract is purified from unbound biotin by size exclusion chromatographyon a PD10-column (Pharmacia). The fraction containing the allergens iscollected. The biotinylated Betula verrucosa is diluted with Phosphatebuffered saline, pH 7.2 (PBS), containing 0.1% human serum albumin(Sigma) and 0.09% NaN₃ (Merck).

Preparation of streptavidin acridinium Ester Label

Streptavidin (Boehringer Mannheim) was conjugated with NSP-DMAE-NHS{2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl-10-(3-sulfopropyl)-acridinium-9-carboxylate}using a modified method or Weeks et al. (ref. 1).

Preparation of Streptavidin-acridiniun Ester Label (lite reagent):

NSP-DMAE-NHS (Chiron Diagnostics, MA, USA) is dissolved inDimethylformamide (Merck) to a final concentration of 1 mg/ml. 0.5 ml ofthis solution is added to 1.7 ml 5.88 mg/ml streptavidin in 0.1 M SodiumPhosphate buffer, 0.15 M NaCl, pH 8.5. The reagents are incubataed for30 minutes at 25° C. with stirring. After incubation 0.4 ml 20 mg/ml6-Amino-n-hexanoic acid (Sigma) is added. To remove unbound DMAE thesolution is loaded onto a PD-10 column (Pharmacia, Uppsala, Sweden). Theconjugated streptavidin is eluted with Phosphate buffered saline, pH 7.2(PBS) and the fraction containing the conjugated streptavidin iscollected. The conjugated streptavidin is diluted to a finalconcentration with Phosphate buffered saline, pH 7.2 (PBS) containing0.1% HSA Sigma), 1% Tween 20 (Merck) and 0.09% NaN₃.

Immobilization of Antibody to Paramagnetic Particles:

6.5 g paramagnetic particles (Chiron Diagnostics, MA, USA) are washed 3times in 650 ml 0.01 M acetate buffer pH 5.5 using magnetic separation.The particles are activated in 6.25% glutaraldehyde (Merck), 0.01 Macetate buffer pH 5.5 for 3 hours at 25° C. The particles are washed 6times in 650 ml acetate buffer pH 5.5. The particles are coupled with1625 mg monoclonal anti-IgE antibody (BioInvent International AB,Sweden) specific against the IgE Fc domain, for 16 hours at 25° C. Theparticles are washed twice in 650 ml 0.01 M phosphate buffer pH 7.4.Blocking of excess of active groups is performed with 1083 mg HSA(Sigma) dissolved in 0.01 M phosphate buffer pH 7.4 for 20 hours at 25°C. The particles are washed in 650 ml 0.01 M phosphate buffer pH 7.4followed by 3 washes in 650 ml 1 M NaCl (Merck). The particles arewashed 3 times in 0.01 M phosphate buffer pH 7.4 followed by a wash with650 ml PBS pH 7.4, 0.1% HSA (Sigma). The particles are resuspended in325 ml and heat treated for 16 hours at 50° C. The particles are washed4 times in 650 ml 0.01 M phosphate pH7.4, 0.1% HSA. The particles areheat treated at 37° C. for 7 days in 650 ml 0.01 M phosphate pH 7.4,0.1% HSA with a buffer exchange on day 3 and day 5. The particles arediluted to a final concentration with Phosphate buffered saline, pH 7.2(PBS) containing 0.1% HSA (Sigma) and 0.09% NaN₃.

Washing Buffer for Assays:

The washing buffer for assays is composed of 200 mM Potassium phosphatebuffer, pH 7.4, 100 mM Potassium chloride, 0.1% Tween 20 and 0.09% NaN₃.All reagents are from Merck.

EXAMPLE 1 Determination of Specific Antibody (specific IgE)

Determination of specific IgE antibodies against Betula verrucosa(Silver birch) allergen was performed by three different methods usingthe reagents described above in the working dilutions defined in eachmethod. The three methods used were Prior Art Method B (FIG. 1b), Method1 according to the invention (FIG. 2a) and Submethod 1 according to theinvention (FIG. 3a).

Prior Art Method B

This method is performed on Ciba Corning ACS:180 Benchtop ImmunoassayAnalyzer described in ref. 2. The analyzer is custom equipped with a 40second time cycle software to give incubation times of two times thenormal. 50 μl of sample and 50 μl of biotinylated Betula verrucosaallergen diluted 1:1500 are dispensed by the sample probe into thecuvette. When the cuvette after 12 minutes of incubation reaches reagentprobe R2, 100 μl of paramagnetic particles diluted 1:20 and 200 μl oflite reagent diluted 1:10000 are dispensed simultaneously and thecuvette moves down the track. The magnets and the wash station arereached after additional 12 minutes and 40 seconds incubation. Wash with750 μl deionized water is performed twice. After completion of the washcycle the paramagnetic particles are resuspended in 300 μl 0.5 g/l H₂O₂in 0.1 M HNO₃. The cuvette enters the luminometer chamber and in frontof the photomultiplier 300 μl 25 mM NaOH solution is added and thephotons of light emitted are measured and quantitated and expressed asrelative light units (RLU). The amount of RLU is proportional to theamount of IgE in the sample. The time from sample dispension to firstresult is 30 minutes and a new result follows every 40 second. Resultswere expressed as RLU experiment/RLU background, where RLU backgroundwas the chemiluminescent reaction in the absence of IgE.

Method 1

This method is performed on a modified version of Ciba Corning ACS:180Benchtop Immunoassay Analyzer described in ref. 2. 25 μl of sample isdispensed by the sample probe into the cuvette and immediately afterthis 100 μl of paramagnetic particles diluted 1:20 is dispensed by afixed probe. After 8 minutes of incubation paramagnetic particles aremagnetically separated and washed once with 1 ml of washing buffer. Theparamagnetic particles are resuspended in 100 μl of washing buffer and50 μl of biotinylated Betula verrucosa allergen diluted 1:1500 is addedto the cuvette. After 10 minutes of incubation, 100 μl of lite reagentdiluted 1:5000 is dispensed with a fixed probe and after additional 8minutes of incubation the paramagnetic particles are magneticallyseparated and washed 3 times with 1 ml of washing buffer. Aftercompletion of the wash cycle the paramagnetic particles are resuspendedin 300 μl 0.5 g/l H₂O₂ in 0.1 M HNO₃. The cuvette enters the luminometerchamber and in front of the photomultiplier 300 μl 25 mM NaOH solutionis added and the photons of light emitted are measured and quantitatedand expressed as relative light units (RLU). The amount of RLU isproportional to the amount of IgE in the sample. Results were expressedas RLU experiment/RLU background, where RLU background was thechemiluminescent reaction in the absence of IgE.

Submethod 1

This method is performed manually in 5 ml polystyrene test tubes(Sarstedt). 25 μl of sample is mixed with 50 μl of biotinylated Betulaverrucosa allergen diluted 1:50. After 12 minutes of incubation at 37°C., 100 μl of paramagnetic particles diluted 1:20 are added and themixture is incubated for 5 minutes and 30 seconds at 37° C. before theparamagnetic particles are magnetically separated and washed once with 3ml of washing buffer using a Magic Lite Multitube Washer (ALK-ASELLÓA/S, Hrsholm, Denmark). The paramagnecic particles are resuspended in200 μl of lite reagent diluted 1:10000 and incubated for 7 minutes and30 seconds at 37° C. before the paramagnetic particles are magneticallyseparated and washed 2 times with 3 ml of washing buffer using a MagicLite Multitube Washer (ALK-ASELLÓ A/S, Hrsholm, Denmark). Theparamagnetic particles are resuspended in 100 μl of deionized water andthe chemiluminescent reaction is measured in a Magic Lite Analyzer II(ALK-ABELLÓ A/S, Hrsholm, Denmark) by adding first 300 μl 0.5 g/l H₂O₂in 0.1 M HNO₃ and then 300 μl 25 mM NaOH solution. The photons of lightemitted are measured and quantitated and expressed as relative lightunits RLU). The amount of RLU is proportional to the amount of IgE inthe sample. Results were expressed as RLU experiment/RLU background,where RLU background was the chemiluminescent reaction in the absence ofIgE.

A serum pool of 5 birch pollen sensitive patients was serially dilutedby a factor two from 140 SU/ml to 1.1 SU/ml to give 8 samples. These 8samples were assayed together with a negative sample (background) and 8samples from birch pollen sensitive individuals in each of the 3 abovedescribed methods, see FIGS. 4, 5 and 6, respectively. It can beconcluded hat all assayed samples can be detected as positive using allthree methods.

EXAMPLE 2 Interference with Specific Antibodies from Other Classes (IgG)

Determination of specific IgE against Betula verrucosa (Silver birch)allergen was performed by the three different methods as described inExample 1 in the presence or varying amounts of competing specificantibodies. More specifically, a serum pool of 5 birch pollen sensitivepatients was mixed in a 1:5 ratio with IgE negative serum containingdifferent titers of rabbit polyclonal anti-Betula verrucosa antibody(ALK-ASELLÓ A/S, Hrsholm, Denmark). The control contained no rabbitantibody.

The results shown in FIG. 7 are expressed as % recovery calculated asthe response obtained with rabbit antibody in the sample relative to thecontrol. Responses were given in Signal/Background. Recoveries less than100% indicate that addition of competing antibody reduces the responsewhile recoveries higher than 100% indicate that addition of competingantibody increases the response.

It can be concluded that Method 1 have less interference with competingantibodies from other classes than the simultaneous method. Whileaddition of competing antibodies results in a significant positiveinterference in Submethod 1, especially with high amounts of interferingantibodies, interference seems to be eliminated completely in Method 1.

EXAMPLE 3 Dilution Curves with Samples from Patients Undergoing SpecificAllergy Vaccination (SAV)

Determination of specific IgE against Alternaria alternata (mould)allergen was Performed by Prior Art Method B and Method 1 as describedin Example 1 except that biotinylated Alternaria alternata allergen wasused instead of biotinylated Betula verrucosa allergen. The workingdilution of biotinylated Alternaria alternata allergen was 1:400 in boththe Prior Art Method and Method 1.

Sera from four patients undergoing Specific Allergy Vaccination (SAV)were serially diluted by a factor two to give four samples prior toanalysis in each of the two different methods. As a reference sample, aserum pool of 5 Alternaria alternate sensitive patients was used.

FIGS. 8-12 show results for samples from the four SAV patients and forthe reference sample. The results shown in FIGS. 8-12 are the responsesobtained with the different diluted sera relative to the expectedresponses. % Recovery is calculated as (Dilution factor)*(Observedresponse)/(Response of undiluted sample). Responses were given inSignal/Background. Recoveries less than 100% indicate that the responsesof the diluted sera are lower than expected while recoveries higher than100% indicate that the responses are higher than expected. Recoverieshigher than 100% therefore indicate that the response obtained with theundiluted sample is an underestimate of the true value.

It appears from FIGS. 8-12 that for the SAV samples the Prior Art Methodunderestimate the true value of the undiluted SAV samples while Method 1result in much more correct estimates. For a serum pool of patients notsubjected to SAV both methods perform satisfactorily by giving much moreaccurate values.

It can be concluded that Method 1 containing 2 washing steps, can muchmore reliably estimate the amount of allergen specific IgE in serumsamples from patients subjected to SAV than the Prior Art Method B.

EXAMPLE 4 Monitoring of Immune Response During House Dust Mite SAV

Determination of specific IgE against Dermatophagoides pteronyssinus(House dust mite) allergen was performed by Prior Art Method B andMethod 1 as described in Example 1 except that biotinylatedDermatophagoides pteronyssinus allergen was used instead of biotinylatedBetula verrucosa allergen. The working dilution of biotinylatedDermatophagoides pteronyssinus allergen was 1:250 in both Prior ArtMethod B and Method 1.

Serum samples were obtained from patients involved in a clinical study.A total of 30 patients were given either placebo treatment (N=14) or SAVwith a 1:1 mixture of Dermatophagoides pteronyssinus andDermatophagoides farinae allergen (Alutard, ALK-ABELLÓ A/S, Hrsholm,Denmark) (N=16). Samples were drawn prior to treatment (t=0), after 6months of treatment (t=6) and after 12 months of treatment (t=12) andall samples were analysed by the two different methods. Results wereobtained as Relativeresponse=(Signal-Background)/(Signal_(high)-Background), whereSignal_(high) is the signal of a serum pool having a high content of thespecific antibody to be detected. These results are shown in FIGS.13-14. One additional set of data was generated by taking the ratiobetween results obtained in the Prior Art Method B and Method 1. Theseresults are shown in FIG. 15.

Conclusion:

Neither of the methods (Prior Art Method B or Method 1) used todetermine serum specific IgE are capable of discriminating active andplacebo treatment groups during SAV (FIGS. 13-14). However, the ratio ofthe results obtained from the Prior Art Method and the method of theinvention (Ratio=IgE (Pior Art Method B)_(t)/IgE (Method 1)_(t))surprisingly discriminates active and placebo treatment groups 6 and 12months after initiation of SAV (FIG. 15). Further, it is likely that aratio cut-off value of approximately 1 may predict if the individualpatient belonged to the placebo or active treatment group (FIG. 17,t=12) i.e. a SAV efficacy parameter.

Statistics:

Statistical analyses were performed by non-parametric tests(Mani-Whitney U-test). All two-sided stochastic probabilities less than0.05 were considered significant. NS signifies non-significant,otherwise the actual probabilities are stated in the Figures.

EXAMPLE 5 Monitoring of Immune Response During House Dust Mite SAV

Determination of specific IgE antibodies against Dermatophagoidespteronyssinus (House dust mite) allergen was performed by two differentmethods using the reagents described above in the working dilutionsdefined in each method. The two methods used were Prior Art Method A(FIG. 1a) and Method 1 according to the invention (FIG. 2a)

Prior Art Method A

This method is performed on a modified version of Ciba Corning ACS:180Benchtop Immunoassay Analyzer described in ref. 3. 25 μl of sample isdispensed by the sample probe into the cuvette and immediately afterthis 100 μl of paramagnetic particles diluted 1:20 is dispensed by afixed probe. After 8 minutes of incubation paramagnetic particles aremagnetically separated and not washed. The paramagnetic particles areresuspended in 100 μl of washing buffer and 50 μl of biotinylatedDermatophagoides pteronyssinus allergen diluted 1:250 is added to thecuvette. After 10 minutes of incubation, 100 μl of lite reagent diluted1:5000 is dispensed with a fixed probe and after additional 8 minutes ofincubation the paramagnetic particles are magnetically separated andwashed 3 times with 1 ml of washing buffer. After completion of the washcycle the paramagnetic particles are resuspended in 300 μl 0.5 g/l H₂O₂in 0.1 M HNO₃. The cuvette enters the luminometer chamber and in frontof the photomultiplier 300 μl 25 mM NaOH solution is added and thephotons of light emitted are measured and quantitated and expressed asrelative light units (RLU). The amount of RLU is proportional to theamount of IgE in the sample. Results were expressed as RLUexperiment/RLU background, where RLU background was the chemiluminescentreaction in the absence of IgE.

Method 1

This method is performed on a modified version of Ciba Corning ACS:180Benchtop Immunoassay Analyzer described in ref. 2. 25 μl of sample isdispensed by the sample probe into the cuvette and immediately afterthis 100 μl of paramagnetic particles diluted 1:20 is dispensed by afixed probe. After 8 minutes of incubation paramagnetic particles aremagnetically separated and washed once with 1 ml of washing buffer.

The paramagnetic particles are resuspended in 100 μl of washing bufferand 50 μl of biotinylated Dermatophagoides pteronyssinus allergendiluted 1:250 is added to the cuvette. After 10 minutes of incubation,100 μl of lite reagent diluted 1:5000 is dispensed with a fixed probeand after additional 8 minutes of incubation the paramagnetic particlesare magnetically separated and washed 3 times with 1 ml of washingbluffer. After completion of the wash cycle the paramagnetic particlesare resuspended in 300 μl 0.5 g/l H₂O₂ in 0.1 M HNO₃. The cuvette entersthe luminometer chamber and in front of the photomultiplier 300 μl 25 mMNaOH solution is added and the photons of light emitted are measured andquantitated and expressed as relative light units (RLU). The amount ofRLU is proportional to the amount of IgE in the sample. Results wereexpressed as RLU experiment/RLU background, where RLU background was thechemiluminescent reaction in the absence of IgE.

The present experimental work involve a total of 28 patients. 13patients were not given any treatment, 7 patients were subjected to SAVtreatment at a dosage level 100, and 8 patients were subjected to SAVtreatment at a dosage level of 300. The SAV treatment consisted inadministering to the patient dosages of Dermatophagoides pteronyssinus(House dust mite) allergen by subcutaneous infections initially duringan up-dosing period of 21 weeks with weekly injections, and subsequentlyduring a two-year period with dosages of 100 or 300 SQ-unitsadministered every 6-8 weeks.

The study uses an effect parameter based upon skin prick test (SPT) andbroncial provocation test (BPT). The clinical effect parameter utilisedhere has been obtained as follows: The relative skin indexes(SI(time=x)−SI(time=0)) were used to cluster the patients in two groups(effect=decrease in skin sensitivity and no effect=unchanged skinsensitivity). Likewise the relative provocation test (logBPT(Time=x)−logBPT(time=0)) were used to cluster the patients in two groups(effect=decreased broncial sensitivity and no effect=unchanged broncialsensitivity). The two measures were combined into one clinical effectparameter: clinical effect=effect in both measures, no clinicaleffect=no effect in both measures and doubtful clinical effect=effect inone of the measures and no effect in the other measure.

FIGS. 16-18 show the relative IgE level at times 0 (LT_S0_(—)0), 6(LT_S6₀), 12 (LT_S12_(—)0) and 24 (LT_S24_(—)0) months for the threeclinical effect groups Effect, No Effect and Doubtful. Time=0 is thepretreatment value. The curve indications having the format nn_iiisignifies patient identification number (nn) and type of treatment(iii), wherein iii=0 means no treatment and iii=100 or 300 meanstreatment at dosage level 100 and 300, respectively. The line at 0.14 isan arbitrary cut off value. The relative IgE level is defined asfollows:

log(Response(Method 1)/Response(Prior Art MethodA))_(t−x)−log(Response(Method 1)/Response(Prior Art Method A))_(t−0),

wherein t=time, x=0, 6, 12 or 24 months and Response=Signal/Background.

As will appear from FIG. 16, the response of Method 1 is higher than theresponse of Prior Art Method A for all of the patients that show aclinical effect, which means that a non IgE competing antibody responsecapable of reacting efficiently with the allergen has been initiated. Itappears from FIG. 17 that none of the patients in the clinical no effectgroup develop a competing antibody response. Finally, it appears fromFIG. 18 that one patient in the clinical doubtful group seem to developa positive competing antibody response which are detected by only skinprick test. The test discriminates between treated and untreatedpatients since no patients from the control group (nn_(—)0) is judged asif they raised a competing antibody response.

Conclusion

The ratio of IgE measured in the absence and presence of competingsubstances in the serum sample, i.e. measured by Method 1 and Prior ArtMethod A, respectively, seems to predict the effect or SAV. Furthermore,the in vitro measured effect is observable early in the treatmentschedule. Although it is probable that the competing agent is an non IgEantibody—any serum substance reacting specifically with the allergenswould be expected to behave similarly.

EXAMPLE 6 Biotinylated Der p

Der p 1 contains only one lysyl residue and the NH2-terminal aminoacidwhich are available for biotinylation. If one biotinylated a crude Der pextract using the “normal” labeling reagents (they target NH2-groups)the resulting labeled Der p extract tend to have biased sensitivitytowards Der p 2 and other allergens.

A more balanced Der p reagent can be obtained as follows: crude Der pextract was biotinylated as described and a preparation of biotinylatedDer p 1, labeled using biotin reagents capable of derivatizing tyrosineand histidine residues, was added. The resulting mixture was used in thefollowing assays.

Biotinylation of Der p 1

Der p 1 (ALK-AbellÓ A/S, Hrsholm, Denmark) was biotinylated withp-Diazobenzoyl Biocytin which was prepared from the stable precursorp-Aminobenzoyl Biocytin (Pierce, USA) according to the manufacturersinstructions. The resulting solution of p-Diazobenzoyl. Biocytin had atheoretical concentration of 1.82 mg/ml (equivalent to 3.38 mM),assuming 100% efficiency of conversion from p-Aminobenzoyl Biocytin top-Diazobenzoyl Biocytin. Der p 1 was biotinylated in a molar ratio of17:1, assuming a molecular weight of Der p 1 of 25 kDa, by adding 343 μlof p-Diazobenzoyl Biocytin solution to 1 ml of 1.74 mg/ml Der p 1 in 0.1M NaHCO₃, pH 8.5. The subsequent steps in the process was as previouslydescribed for the biotinylation with Biotin-XX-NHS ester.

Instrumentation:

The samples were analyzed using an ADVIA Centaur analyzer (BayerDiagnostics). The amount of Der p specific IgE in each sample weredetermined utilizing two protocols: a two step method (T) involvingwashing procedures which remove interfering substances (e.g. non IgEantibodies) before biotinylated allergens are added and a simultaneousprotocol (S) which allow interfering substances (e.g. non IgEantibodies) to be present when the biotinylated allergens are added.Method T and Method S correspond to Method 1 and Prior Art Method Adescribed in Example 5, except that the allergen was biotinylated asdescribed above.

Materials and Methods.

Biotinylated allergen reagent was made by making a 1:250 dilution ofbiotinylated Der p 1 and mixing 1 part of this reagent with 4 parts ofnormal Der p reagent prepared and diluted as previously described.

Serum samples from (N=48) Der p allergic patients receiving SAV with Derp were obtained at 0, 0.5, 1, 2, 3, 6, 9, 12, 18 and 24 month afterinitiation of SAV. The patients were allocated in four treatment groups:controls (N=15), dose_(—)10 (N=12), dose_(—)100 (N=9) and dose_(—)300(N=11) and treated with Der p Alutard for 24 month with the doseindicated.

The clinical parameters SPT (Skin Prick Test), BPT (BronchialProvocation Test) and CPT (Conjunctival Provocation Test, were measuredbefore during and after SAV and a efficacy measure was constructed fromthe cluster analyzed clinical parameters. SAV was judged effective iftwo or three parameters was positive and noneffective if one or none ofthe parameters was positive.

IgE was measured in the serum samples according to the two protocols (Tand S) and a baseline corrected in vitro efficacy parameter wascalculated according to; LN(Ti/Si)LN-(T0/S0) where i is the time(>0<=24) and 0 is the pretreatment values.

Results:

There is a clear dose response relationship between LN(Ti/Si)-LN(T0/S0)and the dose given (FIG. 19, Dose-response relation of the effectparameter) indicating that the parameter measures a dose dependentimmunological change in the patients. FIG. 20 (Mean effect parameter)illustrates the mean values obtained for the in vitro efficacy parameterfor the patients with and without clinical effect and the in vitroparameter is shown for the individual patients in FIG. 21 (Effect:individual patients) and FIG. 22 (No effect: individual patients). Allthe patients with a positive clinical development show an increase ofthe in vitro parameter above the range of the control group from time 6to 24 month and all these patients were receiving an active Der p SAV.Four patients (14%) with no apparent clinical effect are judged to havean effect by the in vitro effect parameter.

Conclusion:

The in vitro effect parameter seems to correlate well with the clinicalassessment of the patient status, and the in vitro parameter ispredictive of the outcome of SAV after 6 month of SAV whereas theclinical effects normally are obvious after 12 or 24 month.

REFERENCES

1) Ian Weeks, Iraj Beheshti, Frank McCapra, Anthony K. Campbell and J.Stuart Woodhead, Clinical Chemistry, 1983, 29, 1474-1479 (1983).

2) J. Boland, G. Carey, E. Tsrodel and M. Kwiatkowski, ClinicalChemistry, 1990, 36, 1598-1602.

What is claimed is:
 1. A method of evaluating immunological status of asubject comprising the steps of: A) determining content of an antibodyin a liquid sample from the subject using the following assay protocol;(Aa) mixing (i) the antibody of the sample, (ii) a reactant antibodydirected against an Fc region of the sample antibody, the reactantantibody being bound to a solid carrier and (iii) a ligand selected froman antigen, an antibody or a hapten, the ligand being directed to an Fabregion of the sample antibody, to form a mixture of a three-componentsolid phase complex and a liquid phase, (Ab) contacting thethree-component complex with (iv) a label compound to form a mixture ofa four-component complex and a liquid phase, (Ac) washing thefour-component solid phase to remove non-complex bound compounds, (Ad)detecting and measuring the washed labeled four-component complex toobtain a measurement A; (B) determining content of said antibody in saidsample using the following assay protocol: (Ba) mixing (i) the antibodyof the sample, and (ii) a reactant antibody directed against the Fcregion of the sample antibody, the reactant antibody being bound to asolid carrier, to form a mixture of a two-component solid phase complexand a liquid phase, (Bb) washing the two-component solid phase complexto remove noncomplex bound compounds, (Bc) contacting the washedtwo-component solid phase complex with a (iii) a ligand selected from anantigen, an antibody or a hapten, the ligand being bound to the Fabregion of the sample antibody, and (iv) a label compound, to form amixture of a four-component solid phase complex and a liquid phase, (Bd)washing the four-component solid phase complex to remove noncomplexbound compounds, (Be) detecting and measuring the washed labeledfour-component complex to obtain a measurement B; and (E) interrelatingmeasurements A and B to express an interference and using theinterference as a parameter for evaluating the immunological status ofthe subject.
 2. The method according to claim 1, wherein step (Ab) iseffected by adding said (iv) label compound to said mixture of athree-component solid phase complex and a liquid phase in step (Aa). 3.The method according to claim 1, wherein step (Ab) is effected bywashing the three-component complex obtained in step (Aa) to removenon-complex bound compounds and by subsequently adding said (iv) labelcompound to the washed three-component complex.
 4. The method accordingto claim 1, wherein step (Bd) is effected by simultaneous incubation ofsaid (iii) ligand and said (iv) label compound with the two-componentcomplex.
 5. The method according to claim 2, wherein step (Bd) iseffected by simultaneous incubation of said (iii) ligand and said (iv)label compound with the two-component complex.
 6. The method accordingto claim 3, wherein step (Bd) is effected by simultaneous incubation ofsaid (iii) ligand and said (iv) label compound with the two-componentcomplex.
 7. The method according to claim 1, wherein initially said(iii) ligand is added to the two-component complex to form athree-component complex, which is then washed to remove non-complexbound (iii) ligand, and then said (iv) label compound is added to form afour-component complex.
 8. The method according to claim 2, whereininitially said (iii) ligand is added to the two-component complex toform a three-component complex, which is then washed to removenon-complex bound (iii) ligand, and then said (iv) label compound isadded to form a four-component complex.
 9. The method according to claim3, wherein initially said (iii) ligand is added to the two-componentcomplex to form a three-component complex, which is then washed toremove non-complex bound (iii) ligand, and then said (iv) label compoundis added to form a four-component complex.
 10. The method according toclaim 1, wherein the label compound is a luminescent label, achemiluminescent label, an enzyme label, a radioactive label, afluorescent label or an absorbance label.
 11. The method according toclaim 2, wherein the label compound is a luminescent label, achemiluminescent label, an enzyme label, a radioactive label, afluorescent label or an absorbance label.
 12. The method according to 3,wherein the label compound is a luminescent label, a chemiluminescentlabel, an enzyme label, a radioactive label, a fluorescent label or anabsorbance label.
 13. The method according to claim 4, wherein the labelcompound is a luminescent label, a chemiluminescent label, an enzymelabel, a radioactive label, a fluorescent label or an absorbance label.14. The method according to claims 4, wherein the label compound is aluminescent label, a chemiluminescent label, an enzyme label, aradioactive label, a fluorescent label or an absorbance label.
 15. Themethod according to claim 1, wherein the (iii) ligand is biotinylated.16. The method according to claim 2, wherein the (iii) ligand isbiotinylated.
 17. The method according to claim 3, wherein the (iii)ligand is biotinylated.
 18. The method according to claim 4, wherein the(iii) ligand is biotinylated.
 19. The method according to claim 5,wherein the (iii) ligand is biotinylated.
 20. The method according toclaim 15, wherein the (iv) label compound is a chemiluminescent compoundcovalently bound to avidin, streptavidin or a functional derivativethereof.
 21. The method according to claim 16, wherein the (iv) labelcompound is a chemiluminescent compound covalently bound to avidin,streptavidin or a functional derivative thereof.
 22. The methodaccording to claim 17, wherein the (iv) label compound is achemiluminescent compound covalently bound to avidin, streptavidin or afunctional derivative thereof.
 23. The method according to claim 18,wherein the (iv) label compound is a chemiluminescent compoundcovalently bound to avidin, streptavidin or a functional derivativethereof.
 24. The method according to claim 19, wherein the (iv) labelcompound is a chemiluminescent compound covalently bound to avidin,streptavidin or a functional derivative thereof.
 25. The methodaccording to claim 1, wherein the subject to be evaluated is undergoingallergy treatment, allergy vaccination treatment or Specific AllergyVaccination (SAV) treatment.
 26. The method according to claim 1,wherein the carrier is a particle.
 27. The method according to claim 26,wherein the carrier is a paramagnetic particle.
 28. The method accordingto claim 1, wherein the sample antibody is a specific IgE.